The specificity of mutation is being studied by recording the spectrum of mutational changes in large groups of E. coli lacI- mutants obtained by DNA sequence analysis. Sequence analysis is facilitated by the recovery of the mutant lacI genes by genetic recombination from F lac onto an M13 vector which carries both the lacI gene and a defective lacZalphagene (M13lacI+Zalpha-): F lacI-Zalpha+ x M131acI-Zalpha+ leads to M131acI-Zalpha+ This recombinant phage can be easily distinguished from its parent by its ability to perform alpha-complementation in the absence of the inducer IPTG. This new procedure has been applied to the production of a spectrum of spontaneous lacI mutations to serve as a baseline for future specificity studies. The spontaneous spectrum is characterized by the occurrence of at least six different classes of mutations. These include (frameshift) hotspot mutations, deletions, base substitutions, ISl insertions, single-base frameshifts, and duplications. The relative frequencies of the non-hotspot events in lacI are taken to be representative of their general importance in spontaneous mutagenesis.